Objective: Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed
paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen
and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues.
We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo
sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging.

Material and Method: Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression
in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma
tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10
non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based
quantification of fluorescence signals for statistical analysis and validation.

Results: We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan
black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in
vascular tissues.

Conclusion: This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein
for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein
targets.

Immunofluorescence (IF) is a sensitive and versatile tool for
histological studies1. However, IF staining of formalinfixed-
paraffin-embedded (FFPE) tissues is not a routinely
used method in clinical pathology due to the associated
autofluorescence. On the other hand, immunoperoxidase
staining of FFPE tissues is a fundamental technique for
diagnostic pathology as well as research, although there are
some major limitations associated with it. The first among
these is the resolution of antigen localization by light
microscopy that remains limited because chromogenic
substrate saturates and precipitates easily. Another issue is
the thickness of the sections that are imaged for analysis2. Sometimes the antigen diffuses prior to fixation,
making localization difficult specially in the case of soluble
proteins like Glial Fibrilliary Acidic Protein (GFAP)3.
All of these conditions hinder evaluation. Additionally, the
conventional staining method is less specific than antigen localization by immunofluorescence, making quantifiable
assessment difficult and therefore inherently subjective4.

Autofluorescence induced interference is one of the major
hitches in IF analysis of FFPE tissues, as it decreases the
signal-to-noise ratio of specific labeling. The greatest source
of autofluorescence in mammalian tissues is artifacts due
to aldehyde fixation along with endogenous fluorophores:
collagen, elastin, lipofuscin, red blood cells, and others.
Vascular tissues contain massive amounts of collagen and
elastin, along with haem, which are typical constituents of
blood vessels5-7, while lipofuscin is known to accumulate
with aging and oxidative stress in various tissues8,9.
Under a fluorescence or confocal microscope, lipofuscin
appears as tiny, dot like structures within the cells, strongly
fluorescing over a wide excitation range of 360-647 nm10,
which overlaps with the commonly used fluorophores and
interferes with specific immuno-fluorescent signals11. In
the present method validation, we describe the optimized indirect IF method on vascular, archival FFPE tissues and
cryo sections.

Glioma (brain tumor), being highly vascular, proliferative
and invasive, was considered for the present study. The
technique was also performed on brain cryo-sections
and non-malignant brain tissue sections along with lung,
kidney and thyroid tissue sections and their results were
compared for reproducibility. Expression of protein human
Telomerase Reverse Transcriptase (hTERT), implicated as
a proliferation marker in various cancers12-14, was used
to verify and replicate the procedure, and quantitatively
analyze and statistically validate the methodology using
fluorescence tagged antibody. Subsequently, expression of
other nuclear and cytoplasmic fluorescein isothiocyanate
(FITC) tagged protein targets, namely High Mobility
GroupA1 (HMGA1), Kinesin Family Member14 (KIF-14)
and Synaptophysin, was also checked on glioma tissues.

The specific labelling of hTERT protein in selected tissuesections
by immuno-florescence immunohistochemistry
(IF-IHC) was performed as per the protocol discussed here.
To start with, slides were cleaned with neutral detergent,
washed in running tap water for 30 min, and further dipped
in 1% acidic ethanol followed by rinsing in deionised water.
The slides were then coated with poly-L-lysine solution
(1:10, diluted in deionised water), then kept overnight at
room temperature. 3 μm sections were overlaid onto the
coated slides and coded. At the beginning of the procedure,
tissue sections were deparaffinized in the oven at 65°C,
followed by immersing in 3 changes of xylene, 5 minutes each. The slides were then dipped in 100% and 95% ethanol
for 2 changes of 5 minutes each, followed by immersing
in 80%, 70% and 50% alcohol respectively, for 5 minutes
each. Finally, slides were rinsed in running tap water for 15
minutes.

Pre-Treatment
Freshly prepared 0.1% sodium borohydride in phosphate
buffer saline (PBS, pH 7.4) was applied to sections for 2
changes, 2.5 minutes each without any in-between wash.
Then 0.5% crystal violet (10% stock in methanol) freshly
diluted in normal saline was laid on the sections for 9
minutes and drained. Lastly, 0.9% NaCl was overlaid on the
tissue, creating a bubble of liquid over the section for three
washes of 5 minutes each.

Epitope Retrieval
Sodium citrate buffer (0.01 M, pH=6) was preheated at
P-high (1350 watt) for 5 minutes, in a microwave oven.
The slides were immersed in this pre-warmed retrieval
solution and the sections boiled for 6 minutes at P-high.
After a pause of 1 minute to lower down the temperature of
retrieval solution, the sections were re-heated at P-40 (540
watt) for 9 minutes. The container was removed to room
temperature and the slides allowed to cool for 20 minutes.
On cooling, the slides were flushed in running tap water for
15 minutes. Excess water was drained-off from the slides
and the sections covered with PBS (pH=7.4), creating a
bubble of buffer over the tissue for 5 minutes.

Troubleshoot
Standardization of protocol was done by immersing the
slides directly in retrieval solution and boiling the sections
for 5 minutes at P-high, P-80 (1080 watt), P-60 (810 watt)
and P-40, which did not give satisfactory florescence signal
intensity.

Permeabilization
The sections were permeabilized by dipping in 0.2% Triton
X-100 in PBS (pH 7.4) for 45 minutes at room temperature.
Slides were rinsed and PBST (0.1% Tween in PBS) dropped
on the tissue, creating a layer of buffer over the section for
5 minutes.

Blocking
70 μl blocking buffer (10% horse serum and 1% BSA in
PBS, pH=7.4) was pipetted onto the sections followed by
incubation in a humidified chamber at room temperature
for 1 h. Excess blocking buffer was drained off from the
slides before proceeding to the next step.

Antigen Localization
70 μl of primary antibody hTERT (1:750 dilutions)
reconstituted in blocking buffer, was applied to the tissue
sections and incubated overnight at 4˚C in a humidified
chamber. After removal from the incubation chamber,
sections were covered with PBST for 5 minutes creating
a bubble and then rinsed with a gentle stream of PBST
(repeated thrice). Then sections were treated with 70 μl host
specific FITC labelled, secondary antibody (1:300 dilution)
re-constituted in PBS (pH 7.4). Sections were incubated for
60 minutes in the dark in a humidified chamber at room
temperature, followed by washing as mentioned for primary
antibody. Counterstaining of the sections was done using
DAPI (5 μg/ mL in PBS, pH 7.4) for 15 minutes in the dark
at RT, and then the slides were washed as described in the
permeabilization step. This procedure was repeated with
each of the other four antibodies on glioma tissue sections.
The specificity of each monoclonal antibody was checked
by western blot before application.

Final Treatment
After the counterstaining procedure, sections were treated
with a freshly prepared 0.1% solution of SBB in 70%
ethanol, for 5 minutes in the dark. Slides were then given
a final wash with PBST as per procedure followed earlier.
Throughout the procedure the tissue was not touched or
allowed to dry.

Mounting
Slides were mounted in the dark using antifade, placed on a
filter paper to drain excess mounting medium and allowed
to dry. The cover-slips were sealed with transparent nail
varnish and the slides were then kept for 1 hr at -20°C for
curing before visualizing.

The slides were stored in tight moisture-free boxes for 18-
24 months and could be reviewed thrice without fading of
fluorescence signal. Each glioma sample was processed in
duplicate sections for the complete procedure.

Quantification of fluorescence signals: Image-J software
generated data on fluorescence signal intensity was analyzed
separately by two different observers. The results were
compared and found to be reproducible. The procedure
adopted for this was as follows: an outline was drawn
around each positively expressing cell, for calculating the
area under consideration, mean fluorescence and integrated
density. This step was repeated for adjacent background by
creating a grid. The corrected total cellular fluorescence
(CTCF) was calculated as = Integrated density - (area of
selected cell × mean fluorescence of background).The
value of CTCF for treated cells was then equalized against
the CTCF of untreated cells in the same field of view and
results compiled for the trio treatment.

In this study, a preliminary screening was done to
determine the efficacy of the selected agents singularly
and in combination(s); and also to define the sequence
that can efficiently quench autofluorescence. Treatment of
copper sulphate in 50 mM sodium acetate for 10 minutes
(Figure 1A-C), ammonium chloride treatment (50 mM in
Tris buffered saline for 10 minutes) (Figure 1B, F) and high
performance UV-C light (15 W/single wavelength 254 nm)
treatment for 3 hours (Figure 1C-G) individually showed
only negligible changes. When a combination of sodium
borohydride and SBB was tried out, partial reduction of
autofluorescent background and non-specific binding
could be achieved (Figure 1D, H).

Figure 1: Superiority of present methodology over some commonly used reagents. FFPE sections A-D) processed as per conventional
IF protocol. Corresponding sections treated with E) CuSO4, F) NH4Cl, G) Single wavelength 254 nm UV, H) Combination treatment
with NaBH4 and SBB; all leading to only fractional reduction of autofluorescent background and non-specific binding. (Autofluorescent
background is highlighted in red circle while lipofuscin and red blood cells are marked by white arrows.) (Scale Bar: 50 μm)

To validate staining integrity following our triple treatment
protocol, we took a two-pronged approach: staining of the tissue specimen by conventional IF protocol (Figure 2A),
and staining of section from the same block with selected
reagents. All test reagent incubations were performed
at RT. For all reagents, various concentrations as well as
incubation periods were tested until a satisfactory result
was obtained or it failed. A single application of 0.1%
sodium borohydride resulted in slight dampening of
autofluorescent background (Figure 2B). 0.5% crystal violet
treatment (Figure 2C) resulted in partial quenching of
background with maximization of both specific and nonspecific
signals. Lone application of 0.1% SBB for 5 minutes
(Figure 2D) eliminated background autofluorescence but
also led to a drastic reduction of both specific and nonspecific
signal intensities. Therefore, sodium borohydride,
crystal violet and SBB, the most promising candidates, were
selected for further study.

Figure 2: Histochemical treatments for reducing tissue autofluorescence. FFPE section A) Processed as per conventional IF protocol,
B) Treated singularly with sodium borohydride resulting only in slight dampening of autofluorescent background, C) Crystal violet
application resulted in partial quenching of autofluorescent background while maximizing both specific and non-specific signals,
D) Application of SBB eliminated lipofuscin autofluorescence, resulting in total diminishing of autofluorescence but also a drastic
reduction of specific and non-specific signal intensities, E) Combined treatment with sodium borohydride + crystal violet + SBB
resulted in total quenching of autofluorescent background, masking of lipofuscin and non-specific signals. Quenching of red blood cell
autofluorescence F) Red blood cells exhibiting intense green fluorescence which interferes with FITC labeled signal of tagged molecule in
the section processed as per conventional IF protocol, G) Complete quenching of blood cell and background autofluorescence after trio
treatment. Lipofuscin and red blood cells marked by white arrows and nonspecific green autofluorescent background highlighted with
a red circle. Immunofluorescence labeled signal after storage of FFPE samples H) Image taken within 24 hours of fluorescent labeling
that is on day 1, I) Section imaged again after storage at -20°C for 24 months clearly indicating that the signal intensity remains the same
(Scale Bar: 50 μm)

Combinational testing for treatment sequence was then
performed to attain the best possible signals for protein
of interest; and to achieve reproducible observations and
imaging. The combination of sodium borohydride, crystal
violet and SBB, in this specific order, resulted in complete
dampening of autofluorescent background, masking of
lipofuscin and non-specific signals; with brighter/ more
intense fluorescent signal of the tagged protein (Figure 2E)
as compared to these treatments alone or a combination
(refer Figure 3 for CTCF values of treatments 2A to 2E).

Dealing with the other element, namely red blood cells,
which exhibit green fluorescence that overlaps in the FITC
channel (as seen in conventionally treated section, Figure
2F); our novel trio treatment resulted in total quenching
of blood vessel autofluorescence as shown in Figure 2G.
Thereafter, the order for this optimal staining combination
was fixed as sodium borohydride followed by crystal violet
treatment before antigen retrieval step and SBB treatment
after staining with DAPI. This proved to be best suited for
diminishment of autofluorescent background and nonspecific
binding; paving way for successful visualization of
the fluorescence tagged marker.

Figure 3: Graph depicts the corrected
total cell fluorescence (CTCF) values of IF
signals of different treatments represented
in Figure 2 A to E and 2 H to I, indicating
that trio treatment is best suited for
complete quenching of autofluorescence
and maximization of signal intensity of
target protein.
NaBH4: Sodium-borohydride,
CV: Crystal violet, SBB: Sudan Black B,
Triple treatment: Combination of NaBH4,
CV and SBB in that order

The increased intensity of desired signal achieved by
our trio treatment has also enabled us to overcome the
limitation of archiving IF labelled sections, facilitating storage for as long as 24 months with periodic review and
without fading of the signal (Figure 2H, 2I; refer to Figure 3
for CTCF value). This is a huge increment on the previously
reported storage period of 9 months (2). The method was
also successfully assessed and validated using 3 other
antibodies, with both nuclear and cytoplasmic expression
(HMGA1, Synaptophysin, KIF- 14) (Figure 4A-C).

The current standardized IF-IHC protocol also has
potential application on varied vascular tissue types as well
as frozen brain sections, with results comparable to archival
tissue (Figure 5). A negative control to check non-specific
binding of secondary antibody was also employed for the
fluorescence tagged molecule. No evidence of secondary
antibody-induced background fluorescence (non-specific
fluorescence signal) was observed for hTERT protein.

Subsequently, the above standardized method was validated
on different tissue samples and controls. The expression of
hTERT was negative in non-malignant brain tissue samples;
while it was up-regulated in different tumors types.

To validate the results, a quantitative analysis of 10
paired glioma tissue sections was carried out. CTCF was
calculated for positive cells and background in each of
these sections. The Wilcoxon signed-rank paired t-test
showed that signal intensity of protein expressing cells in
terms of CTCF of treated sections (median 3261.80 (IQR
1780.44-6075.93)) was significantly higher than CTCF of
untreated sections (median 1164.54 (IQR 574.925-2225.10),
p=0.002). However, the signal intensity of the background
in treated sections was significantly lower (median 235.174 (IQR 46.27-570.32)) than the background of untreated
sections (median 2950.79 (IQR 742.71-5340.73), p=0.002),
validating our methodology (Table I).

IHC is a validated technique for diagnostics and research.
During the use of a chromogenic system for antigen
localization, problems may arise due to saturation and
precipitation of substrate15 or diffusion of the antigen16 making quantification difficult. However, IF-IHC
staining, though more sensitive, is not frequently applied
to FFPE tissues in diagnostic histopathology, the perceived
thought being that endogenous autofluorescence of vascular
tissues renders IF imaging unreliable. In our case as well, efficacious visualization of specific fluorescent label was
hindered by extensive autofluorescence when conventional
IF protocol was employed on different human FFPE tissues
and cryo sections. Therefore, the present study evaluated
protocols for diminishing tissue autofluorescence in
archival and cryo sections from different types of vascular
tissues.

Our reagent selection was led by protocols frequently
reported in the literature for quenching autofluorescence;
hence sodium borohydride, crystal violet, UV-treatment,
SBB, copper sulphate and ammonium chloride were
selected to obtain a reduction in background. Santhosh
Kumar and co-workers have recommended the use of UVlight17. Nybo has employed ammonium chloride to
quench autofluorescence18, while Spitzeret et al. have
suggested the use of copper sulphate with ammonium
acetate for achieving the desired results19.

Sodium borohydride (1% in phosphate buffer) treatment
has been used for both antigen retrieval and to reduce
aldehyde induced autofluorescence of formalin fixed tissue20. Crystal violet treatment of FFPE tissue sections is
cited to reduce the endogenous autofluorescence21 while
allowing IF staining with FITC conjugated antibodies22.
Tissue treatment with 0.1% SBB in 70% ethanol has also
been suggested as a good method to reduce/ eliminate tissue
autofluorescence and background, while preserving the
specific fluorescence signals23,24. In a study by Viegas
et al. a combination of short-duration, high-intensity UV
irradiation and SBB has been suggested as a better approach
to reduce autofluorescence in highly vascular, high
lipofuscin containing murine kidney tissues25. Another
documentation of sequential treatments by Kajimura et al.
advocates the use of 0.1% sodium borohydride followed
by 0.3% SBB in 70% ethanol as an effective procedure for
decreasing autofluorescence26. However, the current
novel trio-treatment of sodium borohydride, crystal violet
and SBB gave best reproducible results in all tissues types
with both nuclear and cytoplasmic antigen localization,
without compromising the signal intensity on extended
storage.

Our study outcome thus clearly indicates that this indirect-
IF based triple treatment methodology is able to provide
successful quantifiable observations with a dampening
of autofluorescence and non-specific background
fluorescence, findings that can be extrapolated to other
vascular human FFPE tissues and cryo sections for different
protein targets.

CONFLICT of INTEREST
The authors declare that there are no competing interests.

ACKNOWLEDGEMENTS
The authors are thankful to M.P. Biotech Council, M.P.
(project no.249) for financial assistance and BMHRC
for infrastructural facilities. The present study is part
of approved project IEC/21/Res/11. The authors kindly
acknowledge Dr. Nitin Garg and Dr. Sandeep K Sorte,
Dept. of Neurosurgery for referral of glioma cases. The
authors are also thankful to Ms. Kavita Niraj for assistance
in literature survey and standardization of protocol, to
Dr. Hunni Gulwani and staff of Histopathology, Dept. of
Pathology, for providing tissue sections.